Studies of the Staphylococcus aureus lytSR regulatory locus have led to the identification of a complex regulatory system that controls the activity of peptidoglycan hydrolases produced by the cell. One component of this regulation is the IrgAB operon whose expression has been shown to inhibit murein hydrolase activity and cause tolerance to penicillin. It has been proposed that the products of this operon function in a manner analogous to bacteriophage-encoded antiholins, which are membrane-associated proteins that inhibit peptidoglycan hydrolase activity at the postranslational level. Recently, homologous proteins encoded by the S. aureus cid operon have been identified and studied. Preliminary experiments indicate that this operon encodes the holin component of this system that enhances peptidoglycan hydrolase activity and increases sensitivity to penicillin. The possibility that the Irg and cid operons are involved in a bacterial programmed cell death (PCD) mechanism has been indicated since the cid mutant exhibits tolerance to other bactericidal agents besides penicillin. These include rifampicin and mitomycin C, which have distinct cellular targets suggesting that this system responds to nonspecific cellular stress by inducing cell death. The recent finding that cid expression is dependent on the alternate stress response sigma factor, Sigma B, strengthens this hypothesis. The experiments described in this proposal are based on four specific aims. The first aim utilizes a genetic approach to explore the role the cid and Irg gene products during the bactericidal response to antibiotics and biocides. The second aim includes flow cytometric studies of membrane potential and cell wall pH designed to more clearly define the roles of the cid and Irg gene products in the regulation of murein hydrolase activity. Purification and analysis of the cid and Irg gene products is the third aim of this proposal with the goal of defining the interactions of these proteins prior to and during cell death. Finally, the fourth aim will study the regulation of cid transcription using molecular strategies to examine the kinetics of cid expression, the cis-acting elements necessary for normal regulation, and the role of a putative transcription activator protein. The long-term objectives of this study are to establish the roles that the Irg and cid operons play in the molecular control of bacterial PCD and to explore novel new therapeutic strategies to combat bacterial infection.